Preparation And Preliminary Application Of Engineering Antibody Against Canine Parvovirus

Canine parvovirus disease is caused by infection with canine parvovirus(CPV),a member of the Parvoviridae.It is a high-risk,high-contact,acute-onset severe infectious disease.The hig h infectivity and high mortality of the disease have caused great economic losses and impacts on the canine and pet breeding industry in our country.At present,anti-canine parvovirus mono clonal antibodies used in clinical treatment are all of mouse origin.When used in dog body tre atment,there are problems that the immune system recognizes and produces canine anti-mouse antibodies,which are quickly cleared.In addition,monoclonal antibody genes are easily lost du ring longer culture,and the method of obtaining antibodies in eukaryotic cells is expensive.Th erefore,in recent years,single-chain antibody(sc Fv)has developed into one of the most studie d genetically engineered antibodies due to its weak immunogenicity and relatively simple and lo w-cost preparation process.In order to obtain a scFv with low cost and therapeutic effect,reduce the clinical treatmen t pressure of canine parvovirus disease,the purpose of this study was to prepare an anti-CPV s cFv using the E.coli expression system,detect its biological activity,and perform preliminary applications.The method is based on the pre-prepared laboratory,which can secrete neutralizing activity of anti-CPV VP2 protein monoclonal antibody hybridoma cell line,extract the total R NA of the hybridoma cells,reverse transcribe and synthesize the cDNA chain,use this as a te mplate to amplify and obtain the antibody heavy chain variable region(VH)gene and light cha in variable region(VL)gene.The variable region gene was connected to the prokaryotic expres sion vector pOPE101,and the recombinant plasmid pOPE-scFv was successfully constructed.Th e sequence of the antibody variable region was obtained by sequencing,VH gene was 387 bp a nd VL gene was 393 bp.Recombinant plasmid transformed XL1-blue competent cells were indu ced by IPTG and expressed by SDS-PAGE and Western blot analysis.The protein was about 35 ku,which was consistent with the expected results.In order to increase the expression level o f scFv,a recombinant plasmid pET23b-GB that simultaneously expressed the antibody variable r egion gene,Erv1 gene and DsbC gene was constructed.After induction of expression by transf ormed Rosetta competent cells,the results of SDS-PAGE and Western blot showed that the exp ression level of scFv was significantly increased and could be recognized by anti-His tag antibo dy.Purification of scFv by affinity chromatography with His-tag resin was 1.120mg/mL.The re sults of enzyme-linked immunosorbent assay(ELISA)showed that the prokaryotic expression of scFv can bind to VP2 protein antigen.Indirect immunofluorescence experiments show that scFv can specifically bind CPV and FPV.The micro-cell neutralization experiment proves that scFv r etains the activity of neutralizing CPV and FPV.The neutralizing titer of CPV is 1:40(0.028 m g/mL)and the neutralizing activity of FPV is 1:20(0.056mg/mL).An animal model of CPV in fection was established,and the diseased dogs recovered after 3 days of continuous injection ofscFv,proving that the prokaryotic expression of scFv can produce a therapeutic and protective effect on CPV infected dogs.To sum up,the variable region sequence of anti-CPV VP2 protein antibody was obtained,and its expression was increased on the basis of achieving prokaryotic expression.The above re search results show that the scFv prepared in this study has the application prospect of treating canine parvovirus disease.